There is a great need for new therapies active in the treatment of viral diseases. Whereas there has been great progress in developing a variety of therapies for the treatment of bacterial infections, there are few viable therapies for the treatment of viruses in general and herpesvirus in particular. Herpesviridae is a family of DNA viruses which include herpes simplex virus type-1 (HSV-1) herpes simplex virus type-2 (HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus, human herpesvirus-6 (HHV6), human herpesvirus-7 (HHV7), pseudorabies and rhinotracheitis, among others. Ganciclovir, acyclovir and foscarnet are currently utilized for the treatment of herpesvirus infections. However, these therapies can have substantial side effects based on their deleterious effects on host cell DNA replication, or they can be used to treat only a limited number of herpesviruses. In addition, herpesviruses are known to develop resistance to these therapies which causes a progressive decline in efficacy [Ljungman et al., J. Infect. Dis., 162, 244 (1990) and Gately et al, J. Infect. Dis., 161, 711 (1990)].
It is known that herpesviruses express their genetic content by directing the synthesis of a number of proteins encoded by the herpesvirus DNA in the host cell. One of the important virus encoded proteins is made as a precursor consisting of an amino terminal-located protease and carboxyl terminal-located assembly protein. This precursor is proteolytically processed in an autocatalytic manner at a specific amino acid sequence known as the "release" site yielding separate protease and assembly protein. The assembly protein is cleaved further by the protease at another specific amino acid sequence known as the "maturation" cleavage site. Recently, EP No. 514,830, published Nov. 25, 1992, describes a virus-specific serine protease which has a role in herpesvirus replication. Additionally, Lui and Roizman (J. Virol, 65, 5149 (1991)) describe the sequence and activity of a protease and the associated assembly protein encoded by U.sub.L 26 of HSV-1. A. R. Welch et al (Proc. Natl. Acad. Sci. USA, 88, 10792 (1991)) describe the related protease (also known as assemblin) and assembly protein encoded by U.sub.L 80 of CMV. An approach currently being investigated for potential use in the treatment of herpesvirus infections is the development of inhibitors of herpesvirus proteases.
Various inhibitors of herpesvirus proteases are described in applicants' copending application.
In order to facilitate the rapid identification of herpesvirus protease inhibitors, an assay which allows for high throughput and linearity is desirable. Initial assays used in the characterization of herpesvirus proteases have been based on electrophoretic separation of products. See EP 514,830. Such method is impractical for screening large numbers of enzymatic inhibitors. An assay which allows for quantitative kinetic characterization of the interaction of the inhibitors with herpesvirus proteases is more preferred.
Resonance energy transfer has been utilized in the biochemical study of hydrolyric enzymes because of the technique's high sensitivity. See e.g. Guilbault, Enzymatic Methods of Analysis, 43-47 (1970).
G. Marshall and M. Toth, in U.S. Pat. No. 5,011,910, describe the use of fluorogenic substrates for the determination of HIV proteases. S. Netzell-Arnett et al (Anal. Biochem., 195, 86 (1991)) describe the use of a fluorescent assay for human matrix metalloproteinases.
C. Garcia-Echeverria and D. Rich describe the use of quenched fluorogenic peptide substrates in an investigation of the sensitivity of the cysteine protease, papain, to modifications in the P1' and P2' sites (FEBS Lett., 297, 100 (1992), and Biochem. and Biophys. Res. Comm., 187, 615 (1992)). E. Matayoshi et al describe the use of an EDANS/DABCYL-containing substrate for assaying HIV protease (Science, 247, 954 (1990)). G. Wang et al use similar groups on their related substrate (Tet. Lett., 31, 6493 (1990)). L. Maggiora et al describe a solid-phase peptide synthesis method of preparing EDANS/DABCYL-containing substrates (J. Med. Chem., 35, 3727 (1992)).